user guide Pichia Expression Kit For expression of recombinant proteins in Pichia pastoris Catalog Number K1710-01 Revision A.0 Publication Number MAN0000012 For Research Use Only. Not for diagnostic procedures. Research Use Only. Not for use in diagnostic procedures. The information in this guide is subject to change without notice. DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. 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All rights reserved. ii Contents Kit contents and storage .................................................................................................................................... 2 Required materials not included with the kit ................................................................................................. 4 Introduction .................................................................................................................... 5 Pichia pastoris expression system .................................................................................................................. 5 Experimental outline .......................................................................................................................................... 8 Methods ........................................................................................................................ 11 Pichia strains ..................................................................................................................................................... 11 E. coli strains ..................................................................................................................................................... 14 Select a Pichia expression vector .................................................................................................................. 15 pHIL-D2 ............................................................................................................................................................... 18 pPIC3.5 ................................................................................................................................................................ 19 pHIL-S1 ............................................................................................................................................................... 20 pPIC9 ................................................................................................................................................................... 21 Signal sequence processing ............................................................................................................................ 22 Clone into the Pichia expression vectors ...................................................................................................... 23 Transformation into E. coli .............................................................................................................................. 29 Prepare transforming DNA ............................................................................................................................. 31 Grow Pichia for spheroplasting ...................................................................................................................... 35 Prepare spheroplasts ...................................................................................................................................... 37 Transform Pichia ............................................................................................................................................... 39 Screen for Mut+ and MutS transformants ...................................................................................................... 42 PCR analysis of Pichia integrants ................................................................................................................... 47 Expression of recombinant Pichia strains .................................................................................................... 49 Analyze samples by SDS-polyacrylamide gel electrophoresis................................................................. 53 Optimize Pichia protein expression ............................................................................................................... 56 Scale up expression .......................................................................................................................................... 58 Protein purification and glycosylation .......................................................................................................... 61 Appendix ....................................................................................................................... 63 E. coli media recipes ......................................................................................................................................... 63 Pichia media recipes ......................................................................................................................................... 64 Proteins expressed in Pichia .......................................................................................................................... 71 Recombination and integration in Pichia ...................................................................................................... 73 Electroporation of Pichia ................................................................................................................................. 77 PEG 1000 transformation method for Pichia ................................................................................................ 78 Lithium chloride transformation method ..................................................................................................... 80 Direct PCR screening of Pichia clones ........................................................................................................... 82 Isolate total DNA from Pichia .......................................................................................................................... 83 Detect multiple integration events ................................................................................................................ 85 Isolate total RNA from Pichia .......................................................................................................................... 88 Beta-Galactosidase assay ................................................................................................................................ 90 Accessory products .......................................................................................................................................... 92 Documentation and support ............................................................................................................................ 93 References ......................................................................................................................................................... 94 Pichia Expression Kit User Guide 1 Kit contents and storage Kit contents The Pichia Expression Kit is shipped at room temperature and contains the following components. Spheroplast Module (Box 1). Store at room temperature. Reagent Amount Components SOS medium 20 mL 1 M Sorbitol 0.3X YPD 10 mM CaCl2 Sterile Water 2 × 125 mL Autoclaved, deionized water SE 2 × 125 mL 1 M Sorbitol 25 mM EDTA, pH 8.0 SCE 2 × 125 mL 1 M Sorbitol 10 mM Sodium citrate buffer, pH 5.8 1 mM EDTA 1 M Sorbitol 2 × 125 mL – CaS 2 × 60 mL 1 M Sorbitol 10 mM Tris-HCl, pH 7.5; 10 mM CaCl2 40% PEG 25 mL 40% (w/v) PEG 3350 (Reagent grade) in water CaT 25 mL 20 mM Tris-HCl, pH 7.5 20 mM CaCl2 Spheroplast Module (Box 2). Store at –20°C. Reagent Amount Components Zymolyase 10 × 20 µL 3 mg/mL Zymolyase in water (100,000 units/g lytic activity) 1 M DTT 10 × 1 mL 1 M dithiothreitol in water Stab Vials: Pichia and E. coli stabs. Store at 4°C. Phenotype Strain Amount Genotype (Pichia only) GS115 1 stab his4 Mut+ KM71 1 stab arg4 his4 aox1::ARG4 MutS, Arg+ GS115 Albumin 1 stab HIS4 MutS GS115 β-Gal 1 stab HIS4 Mut+ TOP10F´ 1 stab F´ {proAB, lacIq, lacZΔM15, Tn10 (TetR)} mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, λ– araD139, Δ(ara- leu)7697, galU, galK, rpsL(StrR), endA1, nupG Continued on next page 2 Pichia Expression Kit User Guide Kit contents and storage, continued Kit contents, Vectors. Store at –20°C. continued Reagent Description pHIL-D2 Vector for intracellular expression in Pichia. 10 µg, 20 µL at 0.5 µg/µL in TE buffer, pH 8.0* pPIC3.5 Vector for intracellular expression in Pichia. 10 µg, 20